The purpose of this research program is twofold. Firstly, to train the candidate in state-of-the-art molecular biological and electrophysiological techniques to study structure-function relationships in ion channels. At the completion of this training program, the applicant will have developed expertise in a number of extremely powerful techniques including patch clamping and single channel analysis, site directed mutagenesis of cDNAs, the polymerase chain reaction, blotting techniques, in vitro mRNA expression, and manipulation and microinjection of mRNA in Xenopus oocytes. The second purpose of this program is to use these techniques to address two important problems in potassium ion channel function. Potassium currents are critical to the maintenance of resting potential and repolarization in a variety of excitable cells, including cardiac cells. K+ channel blockers, which are useful as antiarrhythmic drugs, as well as endogenous neurohumoral agents, such as norepinephrine, regulate K+ channel function and activity. The K+ channel blockers appear to interact with a specific receptor site in the channel, whereas the neurohumoral mediators are believed to alter channel function by phosphorylating specific amino acid residues in the channel protein. Little is known about the structural details of these interactions. Experiments will be performed to determine which amino acids in the channel protein are phosphorylated by specific cellular protein kinases and prove that this phosphorylation causes a specific electrophysiologic change in K+ channel function. Other experiments will determine the primary amino acid structure of the receptor site for various K+ channel blockers in the channel protein and assess how alterations in the receptor site amino acid sequence affects K+ channel conductance and block. Site-specific mutagenesis of K+ channel cDNAs in conjunction with mRNA expression in Xenopus oocytes and patch clamping allows one to study these structure-function relationships.